Fp receptor antagonists or pgf2 alpha antagonists for treating menorrhagia

ABSTRACT

A method of treating or preventing menorrhagia in an female individual the method comprising administering to the individual at least one agent that prevents PGP 2α  having its effect on the FP receptor. Optionally, an inhibitor of PGES and/or an antagonist of EP2 or EP4 is also administered.

The present invention relates to methods of treatment, and in particularmethods of treating menorrhagia.

Menorrhagia is over-abundance of the menstrual discharge.

Menorrhagia affects many women, particularly in the Western world, andrepresent a significant health problem. At least one in 20 women in theUK aged between 34 and 49 years will consult their general practitionersbecause of menstrual problems. These women account for more than one inten of all gynaecological referrals and cost the NHS in excess of £7million per year for medical prescriptions alone. Perceived abnormalvaginal bleeding is said to account for 70% of the at least 70,000hysterectomies done each year.

At present, the treatments used for menorrhagia include tranexamic acidor mefenamic acid. In severe cases the treatment is hysterectomy(vaginal or abdominal) but this is a major operation with seriousmorbidity and some risk of death. A review of treatments for menorrhagiais Stirrat (1999) The Lancet 353, 2175-2176. The development of furtherand alternative therapies is desirable.

The FP prostaglandin receptor has been studied in a variety of tissuesincluding the bovine corpus luteum (Sharif et al 1998, J. Pharmacol.Exp. Ther. 286: 1094-1102); human uterus (Senior et at 1992, Br. J.Pharmacol. 108: 501-506); rabbit jugular vein (Chen et al 1995, Br. J.Pharmacol 116: 3035-3041); various human ocular tissues (Davis & Sharif1999, J. Ocular Pharmacol. Ther. 15: 323-336); and in mouse Swiss 3T3fibroblasts (Griffin et at 1997, J. Pharmacol. Exp. Ther. 281: 845-854);and in rat vascular smooth muscle cells (A7r5) Griffin et at 1998, J.Pharmacol. Exp. Ther. 286: 411-418).

Potent, selective synthetic agonists at some prostaglandin receptorshave been characterised in both in vitro and in vivo models (Coleman etat 1994, Pharmacol. Rev. 46: 205-229). For instance fluprostenol or itsenantiomer (eg AL-5848) (Sharif et al. 1999, J. Pharm. Pharmacol., 51:685-694) and cloprostenol (Coleman et al 1994; Sharif et al 1998) arepotent and selective FP receptor agonists. Since most naturalprostaglandins show rather limited selectivity for their preferredreceptor among this receptor family, the few reported selectiveprostaglandin receptor agonists have been very valuable tools fordiscriminating discrete functional responses coupled to their respectivereceptors. However, conclusive identification of the particularreceptors mediating prostaglandin-stimulated functional responsesrequires potent and selective antagonists (Kenakin 1996, Pharmacol. Rev.48: 413:463).

The recent identification and commercial development of selective FPreceptor agonists as potent and highly efficacious drugs for thetreatment of elevated intraocular pressure (Bito 1997, Surv. Ophthalmol.41 (Suppl. 22): S1-S14; Hellberg et al 1998, Invest. Ophthalmol. Vis.Sci. 39 (Suppl.): 1961) has considerably advanced our knowledge of FPreceptor-coupled pharmacological actions. However, the function of theFP receptor is not fully understood, due in part to significant speciesdifferences in the tissue distribution of this receptor (Ocklind et al1996, Invest. Ophthalmol. Vis. Sci. 37: 716-726; Davis & Sharif 1999;Sharif et al 1999).

Griffin et al (J. Pharmacol. Exp. Ther. 1999, 290: 1278-1284) reportedthe discovery of a selective FP receptor antagonist (AL-8810) ofmicromolar potency. Sharif et al (J. Pharm. Pharmacol. 2000, 52:1529-1539) describe another analogue of PGF_(2α) (AL-3138; Ro-22-6641;11-deoxy-16-fluoro PGF_(2α)) which is a partial agonist of low efficacyand which also functions as an FP receptor antagonist. AL-3138 being arelatively selective agent may be a valuable FP receptor antagonist toolfor investigating the specific function of the FP receptor in variousbiological systems.

Cyclooxygenase (COX) enzymes, also called prostaglandin endoperoxidesynthase (PGHS), catalyse the rate limiting step in the conversion ofarachidonic acid to prostaglandin H₂ (PGH₂). In turn PGH₂ serves as asubstrate for specific prostaglandin synthase enzymes that synthesisethe natural prostaglandins. These are named according to theprostaglandin they produce such that prostaglandin D₂ is synthesised byprostaglandin-D-synthase, prostaglandin E₂ (PGE₂) byprostaglandin-E-synthase (PGES) and prostaglandin F_(2α) (PGF_(2α)) byprostaglandin-F-synthase (PGFS). To date, there are two identifiedisoforms of the COX enzyme, COX-1 and COX-2 (DeWitt, 1991). COX-1 isconstitutively expressed in many tissues and cell types and generatesprostaglandins for normal physiological function (Herschman, 1996). Bycontrast, the expression of COX-2 is rapidly induced followingstimulation of quiescent cells by growth factors, oncogenes, carcinogensand tumour-promoting phorbol esters (Herschman, 1996; Subbaramaiah etal., 1996).

We have shown that expression of the PGF_(2α) receptor in the uterusacross the menstrual cycle demonstrates higher levels of receptorexpression during the proliferative phase of the endometrium comparedwith other stages. Expression in uterine carcinoma tissue issignificantly elevated compared with normal uterine tissue. Using anendometrial epithelial cell line, we have demonstrated that PGF_(2α)induces proliferation of epithelial cells. This proliferation can beinhibited by using specific inhibitors of the PLC signalling pathway. Wehave also shown that the level of FP receptor present in endometrialtissue in women with menorrhagia is greatly increased compared to normaltissue controls.

These observations demonstrate the possibility of antagonising thePGF_(2α) (FP) receptor to combat menorrhagia.

Antagonists of the FP receptor have been suggested for treating orpreventing premature delivery of a foetus and dysmenorrhoea, acting bythe mechanism of relaxation of smooth muscle (WO 99/32640 and WO00/17348). They have not, however, been previously suggested to beuseful in combating menorrhagia.

A first aspect of the invention provides a method of treating orpreventing menorrhagia in a female individual, the method comprisingadministering to the individual at least one agent that preventsPGF_(2α) having its effect on the FP receptor.

It is believed that in some cases menorrhagia may be associated withoverproliferation of the uterine epithelium.

The female individual may be any individual or patient who is sufferingfrom menorrhagia or a patient who is at risk from menorrhagia. Anypremenopausal or perimenopausal woman is at risk of menorrhagia;however, menorrhagia is more common at the beginning and end of awoman's reproductive life so typically there is a greater risk when awoman's periods first start and in women over 40 years of age.

The patient to be treated may be any female individual who would benefitfrom such treatment. Typically and preferably the patient to be treatedis a human female. However, the methods of the invention may be used totreat female mammals, such as the females of the following species:cows, horses, pigs, sheep, cats and dogs. Thus, the methods have uses inboth human and veterinary medicine.

Typically, the agent is one which prevents or disrupts PGF_(2α)-mediatedsignalling of the FP receptor.

Preferably, an agent that prevents PGF_(2α) having its effect on the FPreceptor prevents or reduces the binding of PGF_(2α) to the FP receptor.Alternatively or additionally, the agent may affect the interactionbetween PGF_(2α) and the FP receptor, or the interaction between the FPreceptor and the associated G_(αq) protein, thus inhibiting ordisrupting the PGF_(2α)-FP mediated signal transduction pathway.

In one preferred embodiment, the agent that prevents PGF_(2α) having itseffect on the FP receptor may be an antagonist of the FP receptor. FPreceptor antagonists are typically molecules which bind to the FPreceptor, compete with the binding of the natural ligand PGF_(2α), andinhibit or disrupt the PGF_(2α)-FP mediated signal transduction pathway.In one preferred embodiment, preventing PGF_(2α) having its effect onthe FP receptor includes occupying the PGF_(2α) binding site on theprostaglandin receptor, such that the natural ligand (PGF_(2α)) isprevented from binding in a mode that would result in its normal mode ofsignalling via Gq/Gqπ through inositylphosphate and subsequentmobilisation of intracellular calcium.

Alternatively, the receptor antagonist may be a molecule which binds tothe FP receptor without preventing PGF_(2α) binding thereto, but whichdisrupts the interaction between PGF_(2α) and the FP receptor, thusinhibiting or disrupting PGF_(2α)-FP mediated signal transductionpathway.

Further alternatively, the FP receptor antagonist may be a moleculewhich binds to the FP receptor and which disrupts the interactionbetween the FP receptor and the associated G_(αq) protein, thusinhibiting or disrupting FP mediated signal transduction pathway.

In an alternative preferred embodiment, the agent may be an antagonistof PGF_(2α). PGF_(2α) antagonists are typically molecules which bind toPGF_(2α) and prevent or reduce PGF_(2α) binding to its receptor, whichinhibits or disrupts the PGF_(2α)-FP mediated signal transductionpathway. This is the ‘soluble receptor’ approach in which typicallyeither a part of the receptor or an antibody binds to PGF_(2α).

Alternatively, the PGF_(2α) antagonist may be a molecule which binds toPGF_(2α) without preventing or reducing the binding of PGF_(2α) to theFP receptor, but which disrupts the interaction between PGF_(2α) and theFP receptor such that the PGF_(2α)-FP mediated signal transductionpathway is inhibited or disrupted. This could be a molecule which bindsin a covalent fashion to PGF_(2α) and has no effect on binding potencybut effects the G-protein/IP/Ca²⁺ mechanisms.

In one preferred embodiment, the agent that prevents PGF_(2α) having itseffect on the FP receptor comprises an antagonist of the FP receptor,which may be any FP receptor antagonist that is suitable to beadministered to a patient. The receptor antagonists are typicallyselective to the particular receptor and preferably have an equal orhigher binding affinity to the FP receptor than does PGF_(2α). Althoughantagonists with a higher affinity for the receptor than the naturalligand are preferred, antagonists with a lower affinity may also beused, but it may be necessary to use these at higher concentrations.Preferably, the antagonists bind reversibly to the FP receptor.Preferably, antagonists are selective for a particular receptor and donot affect other receptors; thus, typically, an FP receptor antagonistbinds the FP receptor but does not substantially bind any otherreceptor.

The peptides listed in Table 1 are reported to be antagonists of the FPreceptor that disrupt the interaction between the FP receptor and theassociated G_(60 q) protein (WO 99/32640 and WO 00/17438). The aminoacids are indicated according to the standard IUPAC single letterconvention, and X is cyclohexyl alanine. Lower case letters indicateL-amino acids and capital letters indicate D-amino acids. All of thedisclosure in WO 99/32640 and WO 00/17438 relating to specific peptidesas FP receptor antagonists, is hereby incorporated herein by reference.TABLE 1 PCP-1 RVKFKSQQHRQGRSHHLEM PCP-2RKAVLKNLYKLASQCCGVHVISLHIWELSSIKNSLKVAAIS ESPVAEKSAST PCP-3CLSEEAKEARRINDEIERQLRRDKRDARRE-NH₂ PCP-4 KDTILQLNLKEYNLV-NH₂ PCP-8ilghrdyk PCP-10 wedrfyll PCP-13 ILGHRDYK PCP-14 YQDRFYLL PCP-13.7ILAHRDYK PCP-13.8 ILaHRDYK PCP-13.11 ILGFRDYK PCP-13.13 ILGHKDYKPCP-13.14 ILGHRNYK PCP-13.18 ILGHQDYK PCP-13.20 ILGHRDY-amide PCP-13.21ILGHRDYK-amide PCP-13.22 ILGWRDYK PCP-13.24 ILGXRDYK PCP-15 SNVLCSIF

When the antagonist comprises a peptide, such as those mentioned inTable 1, the antagonist may also comprise protein fusions orpeptidomimetics thereof.

PGF_(2α) dimethyl amide, obtained from Cayman Chemical, Ann Arbor,Mich., USA was reported to be a PGF_(2α) receptor antagonist (Amould etal., (2001) Am. J. Pathol., 159(1): 345-357).

U.S. Pat. No. 6,441,033 B1 (Sharif & Griffin, assigned to AlconManufacturing) describes 11β-fluoro 15β-hydroxy PGF_(2α) analogs whichare FP receptor antagonists.

AL-8810 ((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16, 17, 18,19, 20-pentanor-5,13-prostadienoic acid)obtained from Alcon Research wasreported to be a weak partial agonist of the PGF_(2α) receptor and ahighly selective antagonist of the PGF_(2α) receptor. AL-8810 wasreported not to significantly inhibit functional responses ofprostaglandin receptors TP, DP, EP2 or EP4 at high 10 μM concentration(Griffin et al., (1999) J. Pharmacol. Exp. Ther., 260(3): 1278-1284).

AL-3138 (11-deoxy-16-fluoro PGF_(2α)) was reported to be a weak partialagonist of the PGF_(2α) receptor, and also a highly selective antagonistof the PGF_(2α) receptor (Sharif et al., (2000) J. Pharm. Pharmacol.,52(12): 1529-1539).

Phloretin was reported to be a PGF_(2α) receptor antagonist (Kitanaka etal (1993) J. Neurochem. 60(2): 704-708).

The sulfonylurea glibenclamide was reported to be a PGF_(2α) receptorantagonist (Delaey and Van de Voorde (1995), Eur. J. Pharmacol. 280(2):179-184). The sulfonylureas tolbutamide and tolazamide were reported tobe very weak antagonists of the FP receptor. (Sharif et al (2000) J.Pharm. Pharmacol., 52(12): 1529-1539).

PGF_(2α) dimethyl amine was reported to be a PGF_(2α) receptorantagonist (Stinger et al (1992), J. Pharmacol. Exp. Ther., 220:521-525).

(E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]methylen]amino]oxy]pentanoicacid, also known as ridogrel, obtained from Jannsen Pharmaceutica, wasreported to be a PGF_(2α) receptor antagonist (Jannsens et al., (1990),Thrombosis and Haemostasis, 64(1): 91-96).

The compound PHG113 was reported to be a selective PGF_(2α) receptorantagonist (Quiniou et al., (2001) Pediatric Research, 49(2): 452A.

EP-128479 describes pyrazolyl-methyl-ergoline derivatives which arereported to be PGF_(2α) receptor antagonists. All the disclosure inEP-128479 relating to pyrazolyl-methyl-ergoline derivatives as PGF_(2α)receptor inhibitors, is hereby incorporated herein by reference.

In a further preferred embodiment of the invention, the agent thatprevents PGF_(2α) having its effect on the FP receptor may be anantagonist of PGF_(2α), which may be any PGF_(2α) antagonist that issuitable to be administered to the patient. The PGF_(2α) antagonists arepreferably selective to PGF_(2α) and typically have a higher bindingaffinity for PGF_(2α) than for other molecules. Although antagonistswith a higher affinity for PGF_(2α) than other molecules are preferred,antagonists with a lower affinity may also be used, but it may benecessary to use these at higher concentrations. Preferably, thePGF_(2α) antagonists bind reversibly to PGF_(2α).

PGF_(2α) antagonists include anti-PGF_(2α) antibodies such as rabbitpolyclonal anti-PGF_(2α) antibodies from Oxford Biomedical Research,Inc., Oxford, UK (Arnould et al., Am. J. Pathol. 2001 159(1): 345-357).Amould et al state that, according to the manufacturer, the specificityof the antibody is very high and the cross-reactivity with otherprostanoid derivatives is <1%.

JP 04077480; JP 08176134; JP 01199958; JP 01050818; and JP 63083081 eachdescribe phthalide derivatives that are reported to be PGF_(2α)inhibitors. All the disclosure in JP 04077480; JP 08176134; JP 01199958;JP 01050818; and JP 63083081 relating to phthalide derivatives asPGF_(2α) inhibitors, is hereby incorporated herein by reference.

WO 91/13875 describes (iso) quinoline sulphonamide compounds which arereported to be PGF_(2α) inhibitors. All the disclosure in WO 91/13875relating to (iso) quinoline sulphonamide compounds as PGF_(2α)inhibitors, is hereby incorporated herein by reference.

Some of the compounds reported as being inhibitors or antagonists ofPGF_(2α) may, in fact, be antagonists of the PGF_(2α) (FP) receptor, asused and defined herein. References to such compounds as inhibitors orantagonists of PGF_(2α) should therefore be considered as references toFP receptor antagonists.

As used herein, the term ‘antagonist’ covers all types of antagonism.GPCRs such as prostaglandin receptors are known to show inverse agonismwhich has the outcome of blocking a desired response. Thus a suitable FPantagonist for use in the present invention may be identified bymeasuring the binding of a radio-labelled FP agonist to PGF_(2α) with orwithout the purported antagonist. Secondly, FP antagonists may beidentified in a functional assay eg by showing that the effect of an FPagonist on Ca²⁺ levels is modified in the presence of the antagonist.Thirdly FP antagonists may be identified by inhibition of epithelialcell growth in cell culture.

All of the patents and other documents referred to herein, and inparticular those describing antagonists or inhibitors of FP receptorsand of PGF_(2α), are incorporated herein, in their entirety, byreference.

We have previously found elevated expression of EP2 and EP4 receptors inthe endometrium of menorrhagic women compared to control women (seeExample 3 and PCT/GB02/04845) and have suggested the use of an inhibitorof PGES or an EP2 or EP4 receptor antagonist in the treatment orprevention of menorrhagia (PCT/GB02/004845).

Thus, in a further embodiment of the present invention, in addition tothe at least one agent that prevents PGF_(2α) having its effect on theFP receptor, the individual is also administered one or more of aninhibitor of PGES and/or an antagonist of EP2 or of EP4.

In one embodiment of the invention, the individual is administered aninhibitor of PGES. It has been reported by Thoren & Jakobsson (2000)Eur. J Biochem. 267, 6428-6434 (incorporated herein by reference) thatNS-398, sulindac sulphide and leukotriene C₄ inhibit PGES activity withIC₅₀ values of 20 μM, 80 μM and 5 μM, respectively.

In a still further embodiment of the invention, the individual isadministered an antagonist of an EP2 receptor or an antagonist of an EP4receptor. It will be appreciated that an antagonist of an EP2 receptoror an antagonist of an EP4 receptor is an agent that prevents PGE₂having its effect on the said EP2 or EP4 receptor.

The prostaglandin EP2 receptor antagonist may be any suitable EP2receptor antagonist. Similarly, the prostaglandin EP4 receptorantagonist may be any suitable EP4 receptor antagonist. By “suitable” wemean that the antagonist is one which may be administered to a patient.The receptor antagonists are molecules which bind to their respectivereceptors, compete with the natural ligand (PGE₂) and inhibit theinitiation of the specific receptor-mediated signal transductionpathways. The receptor antagonists are typically selective to theparticular receptor and typically have a higher binding affinity to thereceptor than the natural ligand. Although antagonists with a higheraffinity for the receptor than the natural ligand are preferred,antagonists with a lower affinity may also be used, but it may benecessary to use these at higher concentrations. Preferably, theantagonists bind reversibly to their cognate receptor. Typically,antagonists are selective for a particular receptor and do not affectthe other receptor; thus, typically, an EP2 receptor antagonist bindsthe EP2 receptor but does not substantially bind the EP4 receptor,whereas an EP4 receptor antagonist binds the EP4 receptor but does notsubstantially bind the EP2 receptor. Preferably, the EP2 or EP4 receptorantagonist is selective for the particular receptor subtype. By this ismeant that the antagonist has a binding affinity for the particularreceptor subtype which is at least ten-fold higher than for at least oneof the other EP receptor subtypes. Thus, selective EP4 receptorantagonists have at least a ten-fold higher affinity for the EP4receptor than any of the EP1, EP2 or EP3 receptor subtypes.

It is particularly preferred that the EP2 or EP4 receptor antagonist isselective for its cognate receptor.

EP2 receptor antagonists include AH6809 (Pelletier et al (2001) Br. J.Pharmacol. 132, 999-1008).

EP4 receptor antagonists include AH23848B (developed by Glaxo) andAH22921X (Pelletier et al (2001) Br. J. Pharmacol. 132, 999-1008. Thechemical name for AH23848B is ([1alpha(z),2beta5alpha]-(±)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxo-cyclopentyl]-4-heptenoicacid) (see Hillock & Crankshaw (1999) Eur. J. Pharmacol. 28, 99-108).EP4RA (Li (2000) Endocrinology 141, 2054-61) is an EP(4)-selectiveligand (Machwate et al (2001) Mol. Pharmacol. 60: 36-41). Theomega-substituted 5 prostaglandin E derivatives described in WO 00/15608(EP 1 114 816) (Ono Pharm Co Ltd) bind EP4 receptors selectively and maybe EP4 receptor antagonists.

Peptides described in WO 01/42281 (Hopital Sainte-Justine) eg:IFTSYLECL, IFASYECL, IFTSAECL, IFTSYEAL, ILASYECL, IFRSTDCL, TSYEAL(with 4-biphenyl alanine), TSYEAL (with homophenyl alanine) are alsodescribed as EP4 receptor antagonists, as are some of the compoundsdescribed in WO 00/18744 (Fujisawa Pharm Co Ltd). The5-thia-prostaglandin E derivatives described in WO 00/03980 (EP 1 097922) (Ono Pharm Co Ltd) may be EP4 receptor antagonists.

EP4 receptor antagonists are also described in WO 01/10426 (Glaxo), WO00/21532 (Merck) and GB 2 330 307 (Glaxo).

WO 00/21532 describes the following as EP4 receptor antagonists:

5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt;

5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one;

5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one;

5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one;

5-butyl-2,4-dihydro4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.

GB 2 330 307 describes [1α(Z), 2β, 5α9-(±)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoicacid and[1R[1α(z),2β,5α]]-(−)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoicacid.

WO 00/18405 (Pharmagene) describes the EP4 receptor antagonists AH22921and AH23848 (which are also described in GB 2 028 805 and U.S. Pat. No.4,342,756). WO 01/72302 (Pharmagene) describes further EP4 receptorantagonists, for example those described by reference to, and includedin the general formula (I) shown on page 8 et seq.

In an embodiment, when one or more of an inhibitor of PGES and/or anantagonist of EP2 or of EP4 is administered to a patient, in addition tothe at least one agent that prevents PGF_(2α) having its effect on theFP receptor, the dose of each compound may be the same as would beadministered individually without reference to the other compound.Alternatively and preferably, lower doses may be administered.

All of the patents and other documents referred to herein that describeantagonists or inhibitors of EP2 or EP4 and PGES, are incorporatedherein, in their entirety, by reference.

It will be appreciated that one or more agents that prevent PGF_(2α)having its effect on the FP receptor may be administered to the patient.Optionally, one or more of an inhibitor of PGES and/or an antagonist ofEP2 or of EP4 may also be administered to the patient. These may all beconsidered “treatment agents” of the invention. It will also beappreciated that when more than one treatment agent is administered tothe patient, they may be administered sequentially or in combination.

The treatment agent(s) are administered in an effective amount to combatthe menorrhagia. Thus, the treatment agents may be used to alleviatesymptoms (ie are used palliatively), or may be used to treat thecondition, or may be used prophylactically to prevent the condition. Thetreatment agent may be administered by any suitable route, and in anysuitable form.

Typically, the aforementioned treatment agent(s) for use in theinvention is administered in a quantity and frequency such that aneffective dose is delivered to at least 90% of the FP, and optionallythe EP2 and/or EP4, receptors (ED₉₀). The potency of the molecule(s)would dictate the dose, as would the formulation and route ofadministration.

The aforementioned treatment agents for use in the invention or aformulation thereof may be administered by any conventional methodincluding oral and parenteral (eg subcutaneous or intramuscular)injection. The treatment may consist of a single dose or a plurality ofdoses over a period of time. The dose to be administered is determinedupon consideration of age, body weight, mode of administration, durationof the treatment and pharmacokinetic and toxicological properties of thetreatment agent or agents. The treatment agents are administered at adose (or in multiple doses) which produces a beneficial therapeuticeffect in the patient. Typically, the treatment agents are administeredat a dose the same as or similar to that used when the treatment agentis used for another medical indication. In any event, the dose suitablefor treatment of a patient may be determined by the physician.

Whilst it is possible for a treatment agent of the invention to beadministered alone or in combination with other said treatment agents,it is preferable to present it or them as a pharmaceutical formulation,together with one or more acceptable carriers. The carrier(s) must be“acceptable” in the sense of being compatible with the treatment agentof the invention and not deleterious to the recipients thereof.Typically, the carriers will be water or saline which will be sterileand pyrogen free.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the treatmentagent or agents with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient (ie treatmentagent or agents) with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (eg sodium starch glycolate,cross-linked povidone, cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Moulded tablets may be made bymoulding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethylcellulose in varying proportions to provide desiredrelease profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier. Buccal administration is also preferred.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

Certain of the treatment agents are proteins or peptides. Proteins andpeptides may be delivered using an injectable sustained-release drugdelivery system. These are designed specifically to reduce the frequencyof injections. An example of such a system is Nutropin Depot whichencapsulates recombinant human growth hormone (rhGH) in biodegradablemicrospheres that, once injected, release rhGH slowly over a sustainedperiod.

The protein and peptide can be administered by a surgically implanteddevice that releases the drug directly to the required site. Forexample, Vitrasert releases ganciclovir directly into the eye to treatCMV retinitis. The direct application of this toxic agent to the site ofdisease achieves effective therapy without the drug's significantsystemic side-effects.

Electroporation therapy (EPT) systems can also be employed for theadministration of proteins and peptides. A device which delivers apulsed electric field to cells increases the permeability of the cellmembranes to the drug, resulting in a significant enhancement ofintracellular drug delivery.

Proteins and peptides can be delivered by electroincorporation (EI). EIoccurs when small particles of up to 30 microns in diameter on thesurface of the skin experience electrical pulses identical or similar tothose used in electroporation. In EI, these particles are driven throughthe stratum corneum and into deeper layers of the skin. The particlescan be loaded or coated with drugs or genes or can simply act as“bullets” that generate pores in the skin through which the drugs canenter.

An alternative method of protein and peptide delivery is the ReGelinjectable system that is thermo-sensitive. Below body temperature,ReGel is an injectable liquid while at body temperature it immediatelyforms a gel reservoir that slowly erodes and dissolves into known, safe,biodegradable polymers. The treatment agent is delivered over time asthe biopolymers dissolve.

Protein and peptide pharmaceuticals can also be delivered orally. Theprocess employs a natural process for oral uptake of vitamin B₁₂ in thebody to co-deliver proteins and peptides. By riding the vitamin B₁₂uptake system, the protein or peptide can move through the intestinalwall. Complexes are synthesised between vitamin B₁₂ analogues and thedrug that retain both significant affinity for intrinsic factor (IF) inthe vitamin B₁₂ portion of the complex and significant bioactivity ofthe drug portion of the complex.

Proteins and polypeptides can be introduced to cells by “Trojanpeptides”. These are a class of polypeptides called penetratins whichhave translocating properties and are capable of carrying hydrophiliccompounds across the plasma membrane. This system allows directtargeting of oligopeptides to the cytoplasm and nucleus, and may benon-cell type specific and highly efficient. See Derossi et al (1998),Trends Cell Biol 8, 84-87.

The treatment agents or formulations may also be administeredtransdermally, eg as a patch, gel, lotion, cream or oil.

It is preferred if the treatment agent (or agents) is administeredorally.

It is further preferred if the treatment agent (or agents) isadministered to the female reproductive system. For example, thetreatment agent or agents may suitably be administered intravaginallyusing, for example, a gel or cream or vaginal ring or tampon. Thetreatment agent may also advantageously be administered by intrauterinedelivery, for example using methods well known in the art such as anintrauterine device.

Typically, the gel or cream is one which is formulated foradministration to the vagina. It may be oil based or water based.Typically, the treatment agent (or agents) is present in the cream orgel in a sufficient concentration so that an effective amount isadministered in a single (or in repeated) application.

Typically, the vaginal ring comprises a polymer which formed into a“doughnut” shape which fits within the vagina. The treatment agent (oragents) is present within the polymer, typically as a core, which maydissipate through the polymer and into the vagina and/or cervix in acontrolled fashion. Vaginal rings are known in the art. The vaginal ringmay be disposable and is retained intravaginally during the woman'speriod and therefore contains sufficient of the treatment agent(s) to bereleased and to be effective during the woman's period. Alternatively,the vaginal ring may be used over a time interval of around three monthsto one year, during which time sufficient of the treatment agent(s) isreleased to have a beneficial effect over that period of time. It willbe appreciated that the polymer from which the ring is made, the sizeand shape of the ring and the content of the treatment agent, as well asother parameters, may be selected by reference to whether the ring isfor use in one cycle or for longer spells.

Typically, the tampon is impregnated with the treatment agent (oragents) and that a sufficient amount of the treatment agent (or agents)is present in the tampon.

Typically, the intrauterine device is for placing in the uterus overextended periods of time, such as between one and five years. Typically,the intrauterine device comprises a plastic frame, often in the shape ofa “T” and contains sufficient of the treatment agent(s) to be releasedover the period of use. The agent is generally present within orencompassed by a slow-release polymer which forms part of the device,such as in the form of a “sausage” of agent which wraps around the longarm of the “T” which is typically covered with a controlled-releasemembrane. Intrauterine devices are known in the art.

The invention also provides combinations (such as in a pharmaceuticalformulation) of one or more of the treatment agents as described herein,and one or more agents presently used to treat menorrhagia, such astranexamic acid or mefenamic acid.

A second aspect of the invention provides use of at least one agent thatprevents PGF_(2α) having its effect on the FP receptor, in themanufacture of a medicament for treating or preventing menorrhagia in afemale individual.

It is appreciated that in this and all subsequent aspects of theinvention, preferences for an agent that prevents PGF_(2α) having itseffect on the FP receptor are as described previously with respect tothe first aspect of the invention.

In an embodiment, the female individual is administered one or more ofan inhibitor of PGES and/or an antagonist of EP2 or EP4. Typically thefemale is administered the one or more of these additional agents at thesame time as the medicament. Alternatively, the female may have beenadministered the one or more of these additional agents before receivingthe medicament containing the at least one agent that prevents PGF_(2α)having its effect on the FP receptor. Further alternatively, the femalewill be administered the one or more of these additional agents afterreceiving the medicament containing the at least one agent that preventsPGF_(2α) having its effect on the FP receptor.

It is appreciated that in this and all subsequent aspects of theinvention, preferences for an inhibitor of PGES and/or an antagonist ofEP2 or EP4 are as described previously with respect to the first aspectof the invention.

A third aspect of the invention provides use of a combination of atleast one agent that prevents PGF_(2α) having its effect on the FPreceptor, and one or more of an inhibitor of PGES and/or an antagonistof EP2 or EP4, in the manufacture of a medicament for treating orpreventing menorrhagia in a female individual.

A fourth aspect of the invention provides use of one or more of aninhibitor of PGES and/or an antagonist of EP2 or EP4, in the manufactureof a medicament for treating or preventing a pathological condition ofthe uterus in a female individual, wherein the individual isadministered at least one agent that prevents PGF_(2α) having its effecton the FP receptor. In this case, typically the female is administeredthe at least one agent that prevents PGF_(2α) having its effect on theFP receptor at the same time as the medicament, although the female mayhave been (or will be) administered the at least one agent that preventsPGF_(2α) having its effect on the FP receptor before (or after)receiving the medicament.

A fifth aspect of the invention provides a pharmaceutical compositioncomprising at least one agent that prevents PGF_(2α) having its effecton the FP receptor, for treating or preventing menorrhagia.

Optionally, the pharmaceutical composition further comprises one or moreof an inhibitor of PGES and/or an antagonist of EP2 or EP4.

It is believed that there has been no previous description of acomposition comprising at least one agent that prevents PGF_(2α) havingits effect on the FP receptor, and an inhibitor of PGES and/or anantagonist of EP2 or EP4. Furthermore, it is believed that there hasbeen no previous suggestion that such a composition could be used totreat any medical condition.

Therefore, in a further aspect, the invention includes a compositioncomprising at least one agent that prevents PGF_(2α) having its effecton the FP receptor, and one or more of an inhibitor of PGES and/or anantagonist of EP2 or EP4.

In a yet further aspect, the invention includes a pharmaceuticalcomposition comprising at least one agent that prevents PGF_(2α) havingits effect on the FP receptor, and one or more of an inhibitor of PGESand/or an antagonist of EP2 or EP4, and a pharmaceutically acceptablecarrier.

In a still further aspect, the invention includes a pharmaceuticalcomposition comprising at least one agent that prevents PGF_(2α) havingits effect on the FP receptor, and one or more of an inhibitor of PGESand/or an antagonist of EP2 or EP4, for use in medicine.

Thus the composition is packaged and presented for use in medicine.

The invention will now be described in more detail by reference to thefollowing Figures and Examples.

FIG. 1

In situ hybridisation for FP receptor. Panels a-f are normal humanendometrium in the early-proliferative phase (a), mid-proliferativephase (b), late-proliferative phase (c), early-secretory phase (d),mid-secretory phase (e) and late-secretory phase (f). Panels g-i arepoor (g), moderate (h) and well (i) differentiated samples ofadenocarcinoma tissue.

FIG. 2

Quantitative RT-PCR expression of FP receptor in a) normal humanendometrium and b) adenocarcinoma tissues (p<0.05). In a), EP is earlyproliferative phase, MP/LP is mid/late proliferative phase, ES is earlysecretory phase and LS is late secretory phase. In b), “Cycle” is anaverage of EP, MP/LP, ES and LS from a).

FIG. 3

Total inositol phosphate production in human endometrium (n=2).

FIG. 4

PGF_(2α)-induced ERK phosphorylation in Ishikawa cells. PGF_(2α) 100 nMwas incubated for 10 and 30 min. U73122 2 μM was pre-incubated for 60min before treatment with PGF_(2α).

FIG. 5

PGF_(2α)-induced BrdU incorporation. Proliferation in Ishikawa cellsfollowing PGF_(2α). Cells were treated overnight with PGF_(2α). Allinhibitors were added for 60 min before the addition of PGF_(2α) (n≧4;p<0.05).

FIG. 6

Inmmunocytochemistry of FP receptor expression in human endometriumcollected from women with excessive (A) or normal (B) blood loss. Theinset in B is a negative control. The endometrial tissue from bothmenorrhagic and normal women was collected during the secretory phase ofthe menstrual cycle. In (A), the region of menorrhagic tissue whichstained for expression of the FP receptor is indicated by“}”.

FIG. 7

Endometrial sections from menorrhagic and control women stained withantibodies to the EP2 receptor and EP4 receptor.

EXAMPLE 1 Prostaglandin (PG) F_(2α) Receptor Expression and Localisationin Human Endometrium and Endometrial Adenocarcinoma: Role of PGF_(2α) inEpithelial Cell Proliferation

Summary

PGF_(2α) is one of the prostaglandins generated by human endometrium andcan be measured in menstrual fluid and from endometrial explantscultured in vitro. PGF_(2α) production is stimulated by oestrogen, whichinduces an up-regulation of COX expression, and inhibited byprogesterone, which decreases COX expression and increases PGDHexpression. Increased COX expression has been demonstrated in a numberof different cancers and over-expression in rat intestinal epithelial(RIE) cells induces an altered cell type with increased proliferationand invasiveness in vivo. The aims of this study were to identify thetarget cells for PGF_(2α) in human endometrium and endometrialadenocarcinoma and quantify FP receptor expression in these tissues. Therole of PGF_(2α) in epithelial cell proliferation, and the specificsignalling pathways involved, was then determined in an endometrialadenocarcinoma cell line (Ishikawa). We observed a significantlyincreased expression of FP mRNA in mid- to late-proliferative tissuethat was further increased in endometrial adenocarcinomas. Localisationof FP mRNA was identified in epithelial cells from only mid- andlate-proliferative tissue samples. In uterine adenocarcinoma tissue, FPexpression was consistently localised in epithelial cells and wasindependent of differentiation stage. Negligible FP mRNA expression wasseen in adjacent stromal cells. Inositol mobilisation in response toPGF_(2α) was determined in normal endometrium and endometrialadenocarcinoma and was significantly increased in all samples followingPGF_(2α), however, no additional inositol mobilisation was found inadenocarcinoma samples. In Ishikawa cells, PGF_(2α) also inducedinositol mobilisation and produced a concentration-dependent increase incell proliferation that was inhibited PLC-dependent but not affected byeither p38 or p42/p44 MAPK inhibitors. These results demonstrate thatproliferating endometrial epithelial cells are responsive to PGF_(2α)and indicate a role for PGF_(2α) in menorrhagia.

Introduction

Prostaglandin (PG) F_(2α) is a prostanoid belonging to the eicosanoidfamily of biologically active lipid (1). Other members of thisprostanoid family include PGD₂, PGE₂, prostacyclin (PGI₂) andthromboxane A₂ (TxA₂) that are all synthesised from arachidonic acid bya combination of cyclooxygenase (COX) and specific synthase enzymes (2).To-date, there are two identified isoforms of the COX enzyme;constitutively expressed COX-1 that generates PG for normalphysiological function and COX-2, an early response gene whoseexpression can be rapidly induced (3). COX metabolises arachidonic acidto the unstable intermediate PGH₂ and specific synthase enzymes thenconvert PGH₂ to the PG molecules (4-8). The PG synthase enzymes arenamed according to the prostaglandin they produce such that PGF_(2α) isa metabolite of prostaglandin-F-synthase. Once synthesised PG mediatetheir actions via seven-transmembrane G-protein coupled receptors (GPCR)specific to each prostanoid. PGF_(2α) receptor (FP) has been cloned inhumans and transduces its response via the G-protein Gq, PLC activationand generation of inositol-trisphosphate that in turn mobilisesintracellular Ca²⁺.

COX enzymes and PG have recently been demonstrated to regulateepithelial cell growth and angiogenesis. In rat intestinal epithelialcells, COX-2 expression and PGE₂ synthesis are associated with increasedcellular proliferation and resistance to apoptosis (9,10). The samegroup also showed that expression of COX-2 in epithelial cells enhancesthe expression of angiogenic factors that act in a paracrine manner toinduce endothelial cell migration and microvascular tube formation (9).In human endometrium, COX-2 enzyme expression is maximal during theproliferative phase and is localised to epithelial and perivascularcells (10-14).

PGF_(2α) is also a major metabolite of COX in human endometrium and aswell as being present in menstrual fluid is released by humanendometrial explants in culture (15). However, due to the potentluteolytic activity of PGF_(2α) and the demonstrated increase inmyometrial contractility following PGF_(2α), most studies have focusedon FP receptor expression and regulation in the ovary. FP receptorexpression and the role of PGF_(2α) within the functionalis layer ofhuman endometrium have not been fully examined.

Original studies identified in separated bovine endometrial cellscultured in vitro, that epithelial cells preferentially release PGF_(2α)in contrast to stromal cells that secrete predominately PGE2 (16).Moreover, studies measuring FP mRNA in non-primate species demonstratedincreased FP receptor expression with oestrogen where as progesteronedecreased FP receptor expression when animal were ovariectomised(17-19).

The aims of this study were to localise and quantify FP receptorexpression both in human endometrium and in differing grades of humanuterine adenocarcinoma to identify the PGF_(2α)-responsive cells.Mobilisation of inositol phosphates and phosphorylation of the MAPKextracellular-regulated kinase (ERK) were used to determine the presenceof functional FP receptors and BrdU incorporation in Ishikawa cells asan in-vitro proliferation assay. The data from this study demonstrateepithelial expression of FP in only proliferating tissue withsubstantially increased FP mRNA and clear epithelial cell localisationin endometrial adenocarcinomas. Moreover, PGF_(2α) treatment of Ishikawacells caused inositol mobilisation, ERK phosphorylation andconcentration-dependent increases in cell proliferation.

The data reported herein is the first report of FP expression in humanendometrium and demonstrates a potential role of PGF_(2α) in uterineepithelial cell proliferation and in menorrhagia.

Methods

Patients and Tissue Collection

Endometrial biopsies (n=12) at different stages of the menstrual cyclewere collected with an endometrial suction curette (Pipelle, LaboratoireCCD, France) from women with regular menstrual cycles (25-35 days). Inaddition, full thickness endometrial biopsies (n=18) at all stages ofthe menstrual cycle (n=3 from early, mid and late proliferative and n=3from early mid and late secretory) were collected from women undergoinghysterectomy for benign gynaecological indications. Shortly afterpipelle suction or hysterectomy, tissue was either snap frozen in dryice and stored at −70° C. (for RNA extraction), fixed in neutralbuffered formalin (NBF) and wax embedded (for in-situ hybridisationstudies), or placed in RPMI 1640 (containing 2 mmol/l L-glutamine, 100 Upenicillin and 100 μg/ml streptomycin) and transported to the laboratoryfor in vitro culture. All subjects reported regular menstrual cycles(cycle length 25-35 days) and no women had received a hormonalpreparation in the 3 months preceding biopsy collection. Biopsies weredated according to stated last menstrual period (LMP) and confirmed byhistological assessment according to criteria of Noyes and co-workers(20). Furthermore, circulating oestradiol and progesteroneconcentrations at the time of biopsy were consistent for both stated LMPand histological assignment of menstrual cycle stage. Ethical approvalwas obtained from Lothian Research Ethics Committee and written informedconsent was obtained from all subjects before tissue collection.

In situ Hybridisation (ISH)

Custom synthesis oligonucleotide double FITC-labelled cDNA probes for FPreceptor were obtained from Biognostik GmbH (Germany). Sections (5 μm)were cut onto Gelatin coated Superfrost slides (BDH Laboratory Supplies,UK) from full thickness human uterine biopsies collected across themenstrual cycle (n=18). Tissue was dewaxed in xylene, rehydrated usingincreasing concentrations of ethanol before Proteinase K treatment (100μg/ml in Tris-HCl pH 7.6 100 mM containing EDTA 50 mM) for 15 min at 37°C. to enhance cDNA probe access. After washing in DEPC-H₂O,hybridisation mixture (50 μl; supplied with probe) was added to eachsection and slides incubated for 4 h at 30° C. before adding cDNA probe(6U/ml hybridisation mix) and incubating overnight at 30° C.Post-hybridisation washes of 1×SSC for 5 min (twice) and 0.1×SSC at 42°C. for 15 min (twice) were completed before detecting the FITC-labelledprobe using standard ICC reagents (TSA Biotin System, NEN Life Sciences,UK). Endogenous peroxidase activity was first blocked with 3% H₂O₂ inmethanol for 30 min before incubating sections with blocking buffer for30 min. Conjugated anti-FITC-HRP (Boehringer-Mannheim, Check) was addedin blocking buffer and the sections incubated for 60 min. After washing,biotinyl tyramide amplification reagent was applied to each slide andincubated for 15 min. Streptavidin-HRP was applied after washing andincubated for 30 min and probe localisation visualised with DAB. Controloligonucleotide double FITC-labelled cDNA probe containing the sameproportion of cysteine (C) and guanine (G) bases as the FP receptorprobe was included to assess background hybridisation. All treatmentswere carried out at room temperature unless otherwise specified.

Taqman Quantitative RT-PCR

Endometrial RNA samples were extracted from endometrial biopsies (n=33)using Tri-reagent (Sigma, UTK) following the manufacturer's guidelines.Once extracted and quantified, RNA samples were reverse transcribedusing MgCl₂ (5.5 mM), dNTPs (0.5 mM each), random hexamers (2.5 μM),RNAase inhibitor (0.4 U/μl) and multiscribe reverse transcriptase (1.25U/μl; all from PE Biosystems, Warrington, UK). The mix was aliquotedinto individual tubes (16 μl/tube) and template RNA was added (4 μl/tubeof 100 ng/μl RNA). After mixing by brief centrifugation, samples wereincubated for 90 minutes at 25° C., 45 minutes at 48° C. and 95° C. for5 minutes. Thereafter cDNA samples were stored at −20° C. A tube with noreverse transcriptase was included to control for any DNA contamination.

To measure cDNA expression a reaction mix was prepared containing Taqmanbuffer (5.5 mM MgCl₂, 200 μM dCATP, 200 μM dCTP, 200 μM dGTP, 400 μMdUTP), ribosomal 18S forward and reverse primers and probe (all at 50nM), forward and reverse primers for EP receptor (300 nM), EP receptorprobe (100 nM), AmpErase UNG (0.01 U/μl) and AmpliTaq Gold DNAPolymerase (0.025 U/μl; all from PE Biosystems). After mixing, 48 μl wasaliquoted into separate tubes and 2 μl/replicate (40 ng) of cDNA addedand mixed before placing duplicate 24 μl samples into a PCR plate. A notemplate control (containing water) was included in triplicate. Wellswere sealed with optical caps and the PCR reaction carried out using anABI Prism 7700. FP receptor primers and probe for quantitative PCR weredesigned using the PRIMER express program (PE Biosystems). The sequenceof the FP receptor primers and probe were; Forward 5′-GCA GCT GCG CTTCTT TCA A-3′; Reverse 5′-CAC TGT CAT GAA GAT TAC TGA AAA AAA TAC-3′;Probe (FAM labelled) 5′-CAC AAC CTG CCA GAC GGA AAA CCG-3′.

The ribosomal 18S primers and probe sequences were; Forward 5′-CGG CTACCA CAT CCA AGG AA-3′; Reverse 5′-GCT GGA ATT ACC GCG GCT-3′; Probe (VIClabelled) 5′-TGC TGG CAC CAG ACT TGC CCT C-3′. Data were analysed andprocessed using Sequence Detector vl.6.3 (PE Biosystems) as instructedby the manufacturer. Briefly, the software calculates the reaction cyclenumber at which fluorescence reaches a determined level for both 18Scontrol and FP receptor. This is the relative abundance of FP receptorin each sample and by comparing to an internal positive control,relative expression can be determined. Results are expressed as relativeexpression to the internal positive standard.

Total Inositol Phosphate (InsP) Assays

PGF_(2α) stimulation of total InsP production was as described (21).Briefly, tissue samples or Ishikawa cells were incubated with inositolfree DMEM containing 1% dialyzed heat-inactivated FCS and 0.5 μCi/wellmyo-[³H]inositol (Amersham Pharmacia Biotech) for 48 hours. Medium wasremoved, and cells washed with 1 ml buffer (140 mM NaCl, 20 mM HEPES, 4mM KCl, 8 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂, 1 mg/ml bovine serumalbumin) containing 10 mM LiCl. Cells were then incubated for 1 hour at37° C. in 1 ml buffer with or without inhibitors. Following incubationagonist was added at the required concentration and cells incubated for1 hour. Reactions were terminated by the removal of agonist and theaddition of 500 μl ice cold 10 mM formic acid, which was incubated for30 minutes at 4° C. Total [³H] inositol phosphates was separated fromthe formic acid cell extracts on AG 1-X8 anion exchange resin (Bio-Rad)and eluted with a 1 M ammonium formate/0.1 M formic acid solution. Theassociated radioactivity was determined by liquid scintillation countingand plotted relative to protein concentrations determined using themodified Lowry method.

Proliferation Assay

Proliferation of Ishikawa cells was determined using a BrdUincorporation ELISA (Roche Diagnostics GmbH, Mannheim, Germany).Briefly, Ishikawa cells were seeded at 5×10³ cells per well in a 96-wellplate and allowed to adhere overnight. Cells were next starved for 24 hrwith indomethacin 1.5 μg/ml before 24 h PGF_(2α) treatment (1 nM-1 μM)in serum-free medium containing indomethacin. Inhibitors were added tocells for 60 min before PGF_(2α) with the control well receiving thesame concentration of vehicle. Following 24 h treatment, cells werelabelled with BrdU for 4 h, then fixed and assayed for BrdUincorporation using standard immunohistochemical techniques. Resultswere plotted as percentages of untreated cells.

Statistics

Where appropriate, data were subjected to statistical analysis withANOVA and Fishers PLSD tests (Statview 4.0; Abacus Concepts Inc., USA)and statistical significance accepted when p<0.05.

Results

FP receptor expression in human endometrium demonstrated a distinctivelocalisation pattern across the cycle. FP receptor localised toglandular epithelial cells in only mid- and late-proliferative stages ofthe menstrual cycle and was absent in all other biopsies examined. Onlyoccasional expression was observed in perivascular cells and wasindependent of the cycle stage. In uterine adenocarcinoma biopsies FPreceptor expression was also localised to epithelial cells. Epithelialcell expression of FP receptor was observed in all differentiation typeswith no discernible change in pattern between poor, moderately or welldifferentiated samples.

Quantification of FP receptor mRNA expression in both endometrial andadenocarcinoma samples was determined by Taqman quantitative RT-PCR. Asignificant increase in relative FP receptor expression was observed inmid- to late-proliferative endometrium (0.40±0.02; p≦0.05) when comparedto early proliferative (0.06±0.02) and all secretory phases of themenstrual cycle (0.07±0.01). Within human adenocarcinoma samplesrelative FP receptor mRNA expression was significantly increased(116.3±63.6) compared to cycle endometrium (0.15±0.04). No correlationwas observed between the different grades of adenocarcinoma, however, alarge variance was observed within these tissues.

Human endometrium produced a concentration-dependent increase in totalInsP production following PGF_(2α) treatment. Maximal InsP mobilisationwas observed with PGF_(2α) 100 nM and inhibited by pre-treatment withU73122 2 μM, an inhibitor of PLC. Total inositol phosphate productionwas also measured in Ishikawa cells following treatment with PGF_(2α).PGF_(2α) produced a concentration dependent increase in total InsP withPGF_(2α) 100 nM producing a maximum of 29 cpm/mg protein.

Treatment of Ishikawa cells with PGF_(2α) 100 nM induced phosphorylationof extracellular regulated kinase (ERK) in Ishikawa cells. Treatment for10 min with PGF_(2α) 100 nM induced 6.6±0.7 fold increase inphosphorylated ERK intensity compared to native ERK.

The proliferative effect of PGF_(2α) in Ishikawa cells was determined bymeasuring incorporation of BrdU. PGF_(2α) produced aconcentration-dependent increase in BrdU incorporation that was maximalat 100 nM (136.3±7.48%). Pre-treatment of cells with an ERK1/2 inhibitor(PD98059 50 μM) produced a reduction in basal proliferation (84.0±5.8%)although PGF_(2α) still produced a concentration-dependent increase inproliferation in the presence of PD98059 50 μM (100 nM PGF_(2α)-induced119.4±11.2% control BrdU incorporation). Pre-incubation with aninhibitor of PLCβ (U73122 2 μM) also produced a slight reduction inbasal proliferation (93.9±1.9%), and in addition, inhibited theconcentration-dependent increase in proliferation following PGF_(2α)(106.1±6.6% BrdU incorporation by 100 nM PGF_(2α).

Discussion

We have demonstrated increased expression of FP receptor in epithelialcells from proliferative endometrium and different grades ofadenocarcinoma. Moreover, we have quantified and demonstrated functionalFP receptor expression in Ishikawa cells of human endometrialadenocarcinoma origin. In Ishikawa cells, PGF_(2α) induced IP3production and phosphorylation of p42/p44 MAPK (ERK). Additionally,following overnight incubation with PGF_(2α), Ishiakawa cellsdemonstrated increased cell proliferation that was inhibited by PLCblockade.

PGF_(2α) has not previously been demonstrated to have a role in cellproliferation of mammalian tissues. Studies to identify whether PGF_(2α)was involved in colon adenocarcinomas demonstrated a lack ofproliferation by PGF_(2α) in HCT-8 and HT-29 human colon adenocarcinomacell lines. FP receptor knockout mice do not demonstrate anyabnormalities in endometrial physiology but instead demonstrate failuresin myometrial function with females being unable to deliver pups at term(22).

The data disclosed herein is the first demonstration of theproliferative effects of PGF_(2α) in human endometrial epithelial cells.Maximal FP receptor expression is observed in mid-late proliferativeendometrium when oestrogen levels are elevated.

Without being bound by theory, it believed that the high FP receptorexpression observed in adenocarcinoma samples could relate to levels ofprogesterone receptors where the ratio of PR_(A):PR_(B) may be importantin endometrial cancer (23). In reproductive tissue carcinomas, such asbreast, endometrium and ovary, oestrogens promote cell proliferation andare implicated in disease progression (24). In the endometrium,progesterone opposes the proliferative effect of oestrogen by promotingcell differentiation and progestin treatment has proved beneficial inthe management of endometrial cancer (23, 25). However, therapeuticbenefit is observed only when these tumours express functionalprogesterone receptors (PR) (25) and combining progestins with oestrogenin hormone replacement therapy (HRT), to increase PR expression, hasbeen found to decrease the risk of endometrial cancer (26, 27). As such,loss of responsiveness to progesterone could result in loss ofprogesterone inhibition and explain the observed increase in FP receptorexpression observed in uterine adenocarcinoma.

Ishikawa, an endometrial epithelial cell, expresses greater levels of FPreceptor than normal endometrium and is responsive to PGF_(2α). Weobserved mobilisation of InsP and phosphorylation of ERK in these cellsfollowing PGF_(2α) as has been-previously identified (28, 29). Moreover,PGF_(2α) induced increased proliferation in Ishikawa cells that wassensitive to PLC blockade. This is the first documented report showingepithelial cell proliferation in response to PGF_(2α). Theseobservations support the role of PGF_(2α) in normal epithelial cellproliferation and endometrial adenocarcinoma where FP receptorexpression is increased, and in menorrhagia.

In summary, this is the first demonstration of FP receptor expressionand localisation with human endometrial tissue and human endometrialadenocarcinomas. We have demonstrated epithelial cell expression for FPreceptor and have also demonstrated increased proliferation inendometrial epithelial cells, Ishikawa, following PGF_(2α). Thesestudies clearly indicate a role for PGF_(2α) in epithelial cellproliferation. These studies further suggest that PGF_(2α) has a role inmenorrhagia, and that an agent which prevents PGF_(2α) having its effecton the FP receptor, could be used to combat menorrhagia.

EXAMPLE 2 Expression of FP Receptors in Uterine Tissue of Women withMenorrhagia Compared to Women with no Menorrhagia

Uterine tissue was collected by biopsy from women with a knownindication of menorrhagia and women who have normal uterine function andthe tissue was assessed by immunocytochemistry for the expression of theFP receptor. Briefly, tissue samples were collected, fixed, wax-embeddedand sectioned, and immunohistochemical staining was performed, usingstandard techniques as described in Example 1 of PCT/GB02/04549 and inExample 3 below. The anti-FP receptor antibody used for theimmunocytochemistry was a polyclonal antibody from Cayman Chemicals(distributed by Alexis Corporation Europe, Nottingham, UK, Cataloguenumber 101802), and was used at a 1/50 dilution.

As shown in panel A of FIG. 6, expression of the FP receptor is elevatedin endometrial tissue from women with a known history of menorrhagia,compared to tissue from women with normal blood loss (B). The stainingindicates that the FP receptor is specifically expressed in theglandular epithelial cells of menorrhagic tissue but not of normaltissue.

Hence in women with menorrhagia, it should prove beneficial to treatwith agents that prevent PGF_(2α) having its effect on the FP receptor,such as FP receptor antagonists, in order to block the signallingpathway and ultimately transcription of target genes that may mediatevascular function/dysfunction and excessive bleeding.

EXAMPLE 3 Elevated Expression of EP2 and EP4 Receptors in Endometrium ofMenorrhagic Women Compared to Control Women

Methods

Endometrial sections (5 μm) collected from two women classed as control(with <80 ml blood loss per cycle) or menorrhagic (with >80 ml bloodloss per cycle) were dewaxed in xylene and rehydrated using decreasinggrades of ethanol. After rinsing in PBS, endogenous peroxidase activitywas quenched with 3% H₂O₂ in methanol. Non-immune swine serum (10% serumin PBS) was applied for 20 min before overnight incubation at 4° C. withprimary antibody. An avidin-biotin peroxidase detection system was thenapplied (DAKO Ltd, UK) with 3,3′-diaminobenzidine (DAB) as thechromagen. Sections were counter stained with Harris's haematoxylinbefore mounting. The primary antibodies used in this study were raisedin rabbits against human EP2 or EP4 receptor peptide sequences (CaymanChemicals, USA). The antibody was used at a 1:250 dilution. Alltreatments were carried out at room temperature unless otherwisespecified.

Results

Staining for both EP2 and EP4 receptors was localised in the glandularepithelial cells and endothelial cells. Lower intensity of staining wasobserved in the endometrial samples collected from the woman with normalbleeding pattern as compared with endometrium collected with womensuffering from menorrhagia. This indicates a higher expression patternof the two receptors in the latter group of women.

The results are shown in FIG. 7.

EXAMPLE 4 Treatment of Menorrhagia with an FP Receptor Antagonist

A woman presents to her physician with symptoms of menorrhagia. Thephysician diagnoses menorrhagia. She is administered AL-3138 or AL-8810at a dosing quantity and frequency such that the therapeutic level ofactive agent at the site of treatment is maintained at a level ideallyEC₉₀ but preferably not less than EC₅₀ throughout the treatment period.The treatment is delivered orally or more locally depending on patientacceptability, avoidance of side effects and systemic bioavailability.

EXAMPLE 5 Treatment of Menorrhagia with an FP Receptor Antagonist and anEP2 Receptor Antagonist

A woman presents to her physician with symptoms of menorrhagia. Thephysician diagnoses menorrhagia. She is administered AL-3138 or AL-8810and AH6809 at a dosing quantity and frequency such that the therapeuticlevel of active agents at the site of treatment is maintained at a levelideally EC₉₀ but preferably not less than EC₅₀ throughout the treatmentperiod. The treatment is delivered orally or more locally depending onpatient acceptability, avoidance of side effects and systemicbioavailability.

EXAMPLE 6 Treatment of Menorrhagia with an FP Receptor Antagonist and anEP4 Receptor Antagonist

A woman presents to her physician with symptoms of menorrhagia. Thephysician diagnoses menorrhagia. She is administered AL-3138 or AL-8810and AH23848B at a dosing quantity and frequency such that thetherapeutic level of active agents at the site of treatment ismaintained at a level ideally EC₉₀ but preferably not less than EC₅₀throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 7 Suppository

mg/suppository AL-3138 or AL-8821 (63 μm)* 250 Hard Fat, BP (WitepsolH15 - Dynamit Nobel) 1770 2020*The antagonist AL-3138 or AL-8821 is typically used as a powder whereinat least 90% of the particles are of 63 μm diameter or less.

One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 μm sieve andadded to the molten base with mixing, using a silverson fitted with acutting head, until a smooth dispersion is achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogenous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to 40° C. At a temperature of 38° C. to 40° C. 2.02 gof the mixture is filled into suitable plastic moulds. The suppositoriesare allowed to cool to room temperature.

EXAMPLE 8 Pessaries

mg/pessary AL-3138 or AL-8821 250 Anhydrate Dextrose 380 Potato Starch363 Magnesium Stearate 7 1000

The above ingredients are mixed directly and pessaries prepared bydirect compression of the resulting mixture.

EXAMPLE 9 Vaginal Ring

A vaginal ring containing AL-3138 or AL-8821 is produced using coreextrusion technology.

EXAMPLE 10 Vaginal Ring

A vaginal ring containing AL-3138 or AL-8821 and AH 6809 is producedusing core extrusion technology.

EXAMPLE 11 Intrauterine Device

An intrauterine device containing AL-3138 or AL-8821 is produced usingstandard technology.

EXAMPLE 12 Intrauterine Device

An intrauterine device containing AL-3138 or AL-8821 and AH23848B isproduced using standard technology.

EXAMPLE 13 Tampon

A tampon for treating menorrhagia is produced by impregnating a standardtampon with an effective dose of AL-3138 or AL-8821.

EXAMPLE 14 Tampon

A tampon is produced by impregnating a standard tampon with an effectivedose of AL-3138 or AL-8821 and AH6809 or AH23848B.

REFERENCES FOR EXAMPLE 1

1. Narumiya, S. and FitzGerald, G. A. Genetic and pharmacologicalanalysis of prostanoid receptor function. J Clin Invest, 108: 25-30,2001.

2. Coleman, R. A., Smith, W. L., and Narumiya, S. International Union ofPharmacology classification of prostanoid receptors: properties,distribution, and structure of the receptors and their subtypes.Pharmacol Rev, 46: 205-229, 1994.

3. Vane, J. R., Bakhle, Y. S., and Botting, R. M. Cyclooxygenases 1 and2. Annu Rev Pharmacol Toxicol, 38: 97-120, 1998.

4. Yokoyama, C., Miyata, A., Ihara, H., Ullrich, V., and Tanabe, T.Molecular cloning of human platelet thromboxane A synthase. BiochemBiophys Res Commun, 178: 1479-1484, 1991.

5. Suzuki-Yamamoto, T., Nishizawa, M., Fukui, M., Okuda-Ashitaka, E.,Nakajima, T., Ito, S., and Watanabe, K. cDNA cloning, expression andcharacterization of human prostaglandin F synthase. FEBS Lett, 462:335-340, 1999.

6. Miyata, A., Hara, S., Yokoyama, C., Inoue, H., Ullrich, V., andTanabe, T. Molecular cloning and expression of human prostacyclinsynthase. Biochem Biophys Res Commun, 200: 1728-1734, 1994.

7. Kanaoka, Y., Ago, H., Inagaki, E., Nanayama, T., Miyano, M., Kikuno,R., Fujii, Y., Eguchi, N., Toh, H., Urade, Y., and Hayaishi, O. Cloningand crystal structure of hematopoietic prostaglandin D synthase. Cell,90: 1085-1095, 1997.

8. Forsberg, L., Leeb, L., Thoren, S., Morgenstern, R., and Jakobsson,P. Human glutathione dependent prostaglandin E synthase: gene structureand regulation. FEBS Lett, 471: 78-82, 2000.

9. Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., and DuBois,R. N. Cyclooxygenase regulates angiogenesis induced by colon cancercells. Cell, 93: 705-716, 1998.

10. Van Voorhis, B. J., Huettner, P. C., Clark, M. R., and Hill, J. A.Immunohistochemical localization of prostaglandin H synthase in thefemale reproductive tract and endometriosis. Am J Obstet Gynecol, 163:57-62, 1990.

11. Rees, M. C., Parry, D. M., Anderson, A. B., and Turnbull, A. C.Immunohistochemical localisation of cyclooxygenase in the human uterus.Prostaglandins, 23: 207-214, 1982.

12. Rees, M. C., Anderson, A. B., Demers, L. M., and Turnbull, A. C.Endometrial and myometrial prostaglandin release during the menstrualcycle in relation to menstrual blood loss. J Clin Endocrinol Metab, 58:813-818, 1984.

13. Jones, R. L., Kelly, R. W., and Critchley, H. O. D. Chemokine andcyclooxygenase-2 expression in human endometrium coincides withleukocyte accumulation. Hum Reprod, 12: 1300-1306, 1997.

14. Marions, L. and Danielsson, K. G. Expression of cyclo-oxygenase inhuman endometrium during the implantation period. Mol Hum Reprod, 5:961-965, 1999.

15. Abel, M. H. and Baird, D. T. The effect of 17b-estradiol andprogesterone on prostaglandin production by human endometrium maintainedin organ culture. Endocrinology, 106: 1599-1606, 1980.

16. Fortier, M. A., Guilbault, L. A., and Grasso, F. Specific propertiesof epithelial and stromal cells from the endometrium of cows. J ReprodFertil, 83: 239-248., 1988.

17. Ou, C. W., Chen, Z. Q., Qi, S., and Lye, S. J. Expression andregulation of the messenger ribonucleic acid encoding the prostaglandinF(2alpha) receptor in the rat myometrium during pregnancy and labor. AmJ Obstet Gynecol, 182: 919-925, 2000.

18. Dong, Y. L. and Yallampalli, C. Pregnancy and exogenous steroidtreatments modulate the expression of relaxant EP(2) and contractile FP25 receptors in the rat uterus. Biol Reprod, 62: 533-539, 2000.

19. Hoyer, P. B., Marion, S. L., Stine, I., Rueda, B. R., Hamernik, D.L., Regan, J. W., and Wise, M. E. Ovine prostaglandin F2alpha receptor:steroid influence on steady-state levels of luteal mRNA. Endocrine, 10:105-111, 1999.

20. Noyes, R. W., Hertig, A. T., and Rock, J. Dating the endometrialbiopsy. Fertil Steril, 1: 3-25, 1950.

21. Berg, K. A., Clarke, W. P., Sailstad, C., Saltzman, A., and Maayani,S. Signal transduction differences between 5-hydroxytryptamine type 2Aand type 2C receptor systems. Mol Pharmacol, 46: 477-484, 1994.

22. Sugimoto, Y., Yamasaki, A., Segi, E., Tsuboi, K., Aze, Y., Nishimur,T., Oida, H., Yoshida, N., Tanaka, T., Katsuyama, M., Hasumoto, K.,Murata, T., Hirata, M., Ushikubi, F., Negishi, M., Ichikawa, A., andNarumiya, S. Failure of parturition in mice lacking the prostaglandin Freceptor. Science, 277: 681-683, 1997.

23. Kumar, N. S., Richer, J., Owen, G., Litman, E., Horwitz, K. B., andLeslie, K. K. Selective down-regulation of progesterone receptor isoformB in poorly differentiated human endometrial cancer cells: implicationsfor unopposed estrogen action. Cancer Res, 58: 1860-1865, 1998.

24. Persson, I. Estrogens in the causation of breast, endometrial andovarian cancers—evidence and hypotheses from epidemiological findings. JSteroid Biochem Mol Biol, 74: 357-364, 2000.

25. Sasaki, M., Dharia, A., Oh, B. R., Tanaka, Y., Fujimoto, S., andDahiya, R. Progesterone receptor B gene inactivation and CpGhypermethylation in human uterine endometrial cancer. Cancer Res, 61:97-102, 2001.

26. Dai, D., Kumar, N. S., Wolf, D. M., and Leslie, K. K. Moleculartools to reestablish progestin control of endometrial cancer cellproliferation. Am J Obstet Gynecol, 184: 790-797, 2001.

27. Lim, C. S., Baumann, C. T., Htun, H., Xian, W., Irie, M., Smith, C.L., and Hager, G. L. Differential localization and activity of the A-and B-forms of the human progesterone receptor using green fluorescentprotein chimeras. Mol Endocrinol, 13: 366-375, 1999.

28. Chen, D. B., Westfall, S. D., Fong, H. W., Roberson, M. S., andDavis, J. S. Prostaglandin F2alpha stimulates theRaf/MEK1/mitogen-activated protein kinase signaling cascade in bovineluteal cells. Endocrinology, 139: 3876-3885, 1998.

29. Stocco, C. O., Lau, L. F., and Gibori, G. Acalcium/calmodulin-dependent activation of ERK1/2 mediates JunDphosphorylation and induction of nur77 and 20alpha-hsd genes byprostaglandin F2alpha in ovarian cells. J Biol Chem, 277: 3293-3302,2002.

FURTHER REFERENCES

Ashby, B. (1998) Co-expression of prostaglandin receptors with oppositeeffects: a model for homeostatic control of autocrine and paracrinesignalling. Biochem Pharmacol 55: 239-246.

-   Coleman, R A, Smith, W L, Narumiya, S. (1994) International Union of    Pharmacology classification of prostanoid receptors: properties,    distribution, and structure of the receptors and their subtypes.    Pharmacol Rev 46: 205-229.-   DeWitt, D L. (1991) Prostaglandin endoperoxide synthase: regulation    of enzyme expression. Biochim Biophys Acta 1083: 121-134.-   Herschman, H R. (1996) Prostaglandin synthase 2. Biochim Biophys    Acta 1299: 125-140.-   Subbaramaiah, K, Telang, N, Ramonetti, J T, Araki, R, DeVito, B,    Weksler, B B, Dannenberg, A J. (1996) Transcription of    cyclooxygenase-2 is enhanced in transformed mammary epithelial    cells. Cancer Res 56: 4424-4429.

1. A method of treating or preventing menorrhagia in a femaleindividual, the method comprising administering to the individual atleast one agent that prevents PGF_(2α) having its effect on the FPreceptor.
 2. A method according to claim 1 wherein the agent thatprevents PGF_(2α) having its effect on the FP receptor prevents orreduces the binding of PGF_(2α) to the FP receptor.
 3. A methodaccording to claim 1 wherein the agent that prevents PGF_(2α) having itseffect on the FP receptor affects the interaction between PGF_(2α) andthe FP receptor, or the interaction between the FP receptor and theassociated G_(αq) protein, thus inhibiting or disrupting a PGF_(2α)-FPmediated signal transduction pathway.
 4. A method according to any ofclaim 1 wherein the agent is an antagonist of the FP receptor.
 5. Amethod according to claim 4 wherein the FP receptor antagonist is anyone or more of PGF_(2α) dimethyl amide; PGF_(2α) dimethyl amine; AL-8810((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoicacid); AL-3138 (11-deoxy-16-fluoro PGF_(2α)); phloretin; glibenclamide;ridogrel; PHG113; PCP-1 (rvkfksqqhrqgrshhlem); PCP-2(rkavlknlyklasqccgvhvislhiwelssiknslkvaaisespvaeksast); PCP-3(clseeakearrindeierqlrrdkrdarre-NH₂); PCP-4 (kdtilqlnlkeynlv-NH₂); PCP-8(ilghrdyk); PCP-10 (wedrfyll); PCP-13 (ILGHRDYK); PCP-14 (YQDRFYLL);(ILAHRDYK); PCP-13.7 (ILAHRDYK); PCP-13.8 (ILaHRDYK); PCP-13.11(ILGFRDYK); PCP-13.13 (ILGHKDYK); PCP-13.14 (ILGHRNYK); PCP-13.18(ILGHQDYK); PCP-13.20 (ILGHRDY-amide); PCP-13.21 (ILGHRDYK-amide);PCP-13.22 (ILGWRDYK); PCP-13.24 (ILGXRDYK); and PCP-15 (SNVLCSIF).
 6. Amethod according to claim 1 wherein the agent is an antagonist ofPGF_(2α).
 7. A method according to claim 6 wherein the PGF_(2α)antagonist is an anti-PGF_(2α) antibody.
 8. A method according to claim1 further comprising administering to the individual one or more of aninhibitor of PGES and/or an antagonist of EP2 or EP4.
 9. A methodaccording to claim 8 wherein the antagonist of EP2 or EP4 is AH6809, anomega-substituted prostaglandin E derivative, AH23848B, AH22921X,IFTSYLECL, IFASYECL, IFTSAECL, IFTSYEAL, ILASYECL, IFTSTDCL, TSYEAL(with 4-biphenylalanine), TSYEAL (with homophenylalanine), a5-thia-prostaglandin E derivative,5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt,5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,or5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.10. Use of at least one agent that prevents PGF_(2α) having its effecton the FP receptor, in the manufacture of a medicament for treating orpreventing menorrhagia in a female individual.
 11. Use according toclaim 10, wherein the individual is administered one or more of aninhibitor of PGES and/or an antagonist of EP2 or EP4.
 12. Use of acombination of at least one agent that prevents PGF_(2α), having itseffect on the FP receptor, and one or more of an inhibitor of PGESand/or an antagonist of EP2 or EP4, in the manufacture of a medicamentfor treating or preventing menorrhagia in a female individual.
 13. Useof one or more of an inhibitor of PGES and/or an antagonist of EP2 orEP4 in the manufacture of a medicament for treating or preventingmenorrhagia in a female individual, wherein the female individual isadministered at least one agent that prevents PGF_(2α) having its effecton the FP receptor.
 14. A pharmaceutical composition comprising at leastone agent that prevents PGF_(2α) having its effect on the FP receptorfor treating or preventing menorrhagia in a female individual.
 15. Apharmaceutical composition according to claim 14 further comprising oneor-more of an inhibitor of PGES and/or an antagonist of EP2 or EP4. 16.A vaginal ring or a tampon or an intrauterine device comprising at leastone agent that prevents PGF_(2α) having its effect on the FP receptor.17. A vaginal ring or a tampon or an intrauterine device according toclaim 16 further comprising one or more of an inhibitor of PGES and/oran antagonist of EP2 or EP4.
 18. A use according to claim 10, whereinthe agent that prevents PGF_(2α) having its effect on the FP receptor(i) prevents or reduces the binding of PGF_(2α) to the FP receptor, (ii)affects the interaction between PGF_(2α) and the FP receptor, or theinteraction between the FP receptor and the associated G_(αq) protein,thus inhibiting or disrupting a PGF_(2α)-FP mediated signal transductionpathway, (iii) is an antagonist of the FP receptor, (iv) is anantagonist of PGF_(2α), or (v) is an anti-PGF_(2α) antibody.
 19. Useaccording to claim 11, wherein the antagonist of EP2 or EP4 is selectedfrom the group of AH6809, an omega-substituted prostaglandin Ederivative, AH23848B, AH22921X, IFTSYLECL, IFASYECL, IFTSAECL, IFTSYEAL,ILASYECL, IFTSTDCL, TSYEAL (with 4-biphenylalanine), TSYEAL (withhomophenylalanine), a 5-thia-prostaglandin E derivative,5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt,5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,or5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.20. A composition comprising at least one agent that prevents PGF_(2α)having its effect on the FP receptor, and one or more of an inhibitor ofPGES and/or an antagonist of EP2 or EP4.
 21. A pharmaceuticalcomposition comprising at least one agent that prevents PGF_(2α) havingits effect on the FP receptor, and one or more of an inhibitor of PGESand/or an antagonist of EP2 or EP4, and a pharmaceutically acceptablecarrier.
 22. A composition according to claim 20 for use in medicine.23. Use according to claim 10 wherein the agent that prevents PGF_(2α)having its effect on the FP receptor is an antagonist of the FP receptoris selected from the group of any one or more of PGF_(2α) dimethylamide; PGF_(2α) dimethyl amine; AL-8810((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoicacid); AL-3138 (11-deoxy-16-fluoro PGF_(2α)); phloretin; glibenclamide;ridogrel; PHG113; PCP-1 (rvkfksqqhrqgrshhlem); PCP-2(rkavlknlyklasqccgvhvislhiwelssiknslkvaaisespvaeksast); PCP-3(clseeakearrindeierqlrrdkrdarre-NH₂); PCP-4 (kdtilqlnlkeynlv-NH₂); PCP-8(ilghrdyk); PCP-10 (wedrfyll); PCP-13 (ILGHRDYK); PCP-14 (YQDRFYLL);(ILAHRDYK); PCP-13.7 (ILAHRDYK); PCP-13.8 (ILaHRDYK) ; PCP-13.11(ILGFRDYK); PCP-13.13 (ILGHKDYK); PCP-13.14 (ILGHRNYK); PCP-13.18(ILGHQDYK); PCP-13.20 (ILGHRDY-amide); PCP-13.21 (ILGHRDYK-amide);PCP-13.22 (ILGWRDYK); PCP-13.24 (ILGXRDYK); and PCP-15 (SNVLCSIF).
 24. Apharmaceutical composition according to claim 14 wherein the agent thatprevents PGF_(2α) having its effect on the FP receptor (i) prevents orreduces the binding of PGF_(2α) to the FP receptor, (ii) affects theinteraction between PGF_(2α) and the FP receptor, or the interactionbetween the FP receptor and the associated G_(αq) protein, thusinhibiting or disrupting a PGF_(2α)-FP mediated signal transductionpathway, (iii) is an antagonist of the FP receptor, (iv) is anantagonist of PGF_(2α), or (v) is an anti-PGF_(2α) antibody.
 25. Apharmaceutical composition according to claim 14 wherein the agent thatprevents PGF_(2α) having its effect on the FP receptor is an antagonistof the FP receptor is selected from the group of any one or more ofPGF_(2α) dimethyl amide; PGF_(2α) dimethyl amine; AL-8810((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoicacid); AL-3138 (11-deoxy-16-fluoro PGF_(2α)); phloretin; glibenclamide;ridogrel; PHG113; PCP-1 (rvkfksqqhrqgrshhlem); PCP-2(rkavlknlyklasqccgvhvislhiwelssiknslkvaaisespvaeksast); PCP-3(clseeakearrindeierqlrrdkrdarre-NH₂); PCP-4 (kdtilqlnlkeynlv-NH₂); PCP-8(ilghrdyk); PCP-10 (wedrfyll); PCP-13 (ILGHRDYK); PCP-14 (YQDRFYLL);(ILAHRDYK); PCP-13.7 (ILAHRDYK); PCP-13.8 (ILaHRDYK) ; PCP-13.11(ILGFRDYK); PCP-13.13 (ILGHKDYK); PCP-13.14 (ILGHRNYK); PCP-13.18(ILGHQDYK); PCP-13.20 (ILGHRDY-amide); PCP-13.21 (ILGHRDYK-amide);PCP-13.22 (ILGWRDYK); PCP-13.24 (ILGXRDYK); and PCP-15 (SNVLCSIF).
 26. Avaginal ring or a tampon or an intrauterine device according to claim 16wherein the at least one agent that prevents PGF_(2α) having its effecton the FP receptor (i) prevents or reduces the binding of PGF_(2α) tothe FP receptor, (ii) affects the interaction between PGF_(2α) and theFP receptor, or the interaction between the FP receptor and theassociated G_(α)q protein, thus inhibiting or disrupting a PGF_(2α)-FPmediated signal transduction pathway, (iii) is an antagonist of the FPreceptor, (iv) is an antagonist of PGF_(2α), or (v) is an anti-PGF_(2α)antibody.
 27. A vaginal ring or a tampon or an intrauterine deviceaccording to claim 16 wherein the agent that prevents PGF_(2α) havingits effect on the FP receptor is an antagonist of the FP receptor isselected from the group of any one or more of PGF_(2α) dimethyl amide;PGF_(2α) dimethyl amine; AL-8810 ((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoicacid); AL-3138 (11-deoxy-16-fluoro PGF_(2α)); phloretin; glibenclamide;ridogrel; PHG113; PCP-1 (rvkfksqqhrqgrshhlem); PCP-2(rkavlknlyklasqccgvhvislhiwelssiknslkvaaisespvaeksast); PCP-3 (clseeakearrindeierqlrrdkrdarre-NH₂); PCP-4 (kdtilqlnlkeynlv-NH₂); PCP-8(ilghrdyk); PCP-10 (wedrfyll); PCP-13 (ILGHRDYK); PCP-14 (YQDRFYLL);(ILAHRDYK); PCP-13.7 (ILAHRDYK); PCP-13.8 (ILaHRDYK); PCP-13.11(ILGFRDYK); PCP-13.13 (ILGHKDYK); PCP-13.14 (ILGHRNYK); PCP-13.18(ILGHQDYK); PCP-13.20 (ILGHRDY-amide); PCP-13.21 (ILGHRDYK-amide);PCP-13.22 (ILGWRDYK); PCP-13.24 (ILGXRDYK); and PCP-15 (SNVLCSIF).
 28. Apharmaceutical composition according to claim 15 wherein the antagonistof EP2 or EP4 is selected from the group of AH6809, an omega-substitutedprostaglandin E derivative, AH23848B, AH22921X, IFTSYLECL, IFASYECL,IFTSAECL, IFTSYEAL, ILASYECL, IFTSTDCL, TSYEAL (with 4-biphenylalanine),TSYEAL (with homophenylalanine), a 5-thia-prostaglandin E derivative,5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt,5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,or5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.29. A vaginal ring or a tampon or an intrauterine device according toclaim 17 wherein the antagonist of EP2 or EP4 is selected from the groupof AH6809, an omega-substituted prostaglandin E derivative, AH23848B,AH22921X, IFTSYLECL, IFASYECL, IFTSAECL, IFTSYEAL, ILASYECL, IFTSTDCL,TSYEAL (with 4-biphenylalanine), TSYEAL (with homophenylalanine), a5-thia-prostaglandin E derivative,5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt,5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,or5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.